Myostatin is a transforming growth factor-β (TGF-β) superfamily member that serves as a very potent autocrine/paracrine inhibitor of muscle growth (A. C. McPherron, A. M. Lawler, S. J. Lee, Nature. 387, 83-90, 1997). Myostatin is composed of 376 amino acids, and its precursor protein is activated by 2 cleavages using proteinases. A first cleavage step is to remove a 24-amino acid signal peptide using a purine family of enzymes and a second cleavage step is performed by cleavage by BMP1/Tolloid matrix metalloproteinases. In this case, the cleavage occurs at an Arg-Ser-Arg-Arg (RSRR) site at 240th to 243rd amino acids to generate an N-terminal myostatin propeptide (27.64 kDa) and a C-terminal fragment (12.4 kDa) (S. J. Lee, Annu. Rev. Cell. Dev. Biol. 20, 61-86, 2004). It was known that active types of mature myostatin form dimers through disulfide bonds at the C-terminal region, and shows a 100% homology with those from a mouse, a rat, a pig, a chicken, a turkey, a dog, and the like.
When myostatin is not expressed in mouse cell, a rapid increase in mass of skeletal muscles results in muscle hypertrophy and hyperplasia (A. C. McPherson et al., Nature. 387, 83-90, 1997; J. Lin et al., Biochem. Biophys. Res. Commun. 291, 701-706, 2002; T. A. Zimmers et al. Science. 296, 1486-1488, 2002). In addition to the mice, it was reported that mutations of myostatin in some cattle and sheep results in muscle hypertrophy (G. Hadjipavlou, et. al., Anim. Genet. 39, 346-353, 2008; A. C. McPherson and S. J. Lee, Proc. Natl. Acad. Sci. 94, 12457-12461, 1997; R. Kambadur et al., Genome Res. 7, 910-916, 1997). In recent years, it was reported that myostatin acts by directly binding to activin receptor type IIB (AVR2B) (S. J. Lee, and A. C. McPherron, Proc. Natl. Acad. Sci. U.S.A. 98, 9306-9311, 2001; A. Rebbapragada et al., Mol. Cell. Biol. 23, 7230-7242, 2003; R. S. Thies et al., Growth Factors. 18, 251-259, 2001), and has signaling mechanisms through Smads signaling pathways (S. J. Lee, and A. C. McPherson, Proc. Natl. Acad. Sci. U.S.A. 98, 9306-9311, 2001; A. Rebbapragada et al., Mol. Cell. Biol. 23, 7230-7242, 2003; X. Zhu et al., Cytokine. 26, 262-272, 2004). It was also reported that myostatin affects a p38 MAPK signaling pathway, an Ras-ERK1/2 pathway and a JNK signaling pathway in addition to the Smads signaling pathway (Z. Q. Huang et al., Cell. Signal. 19, 2286-2295, 2007; B. Philip et al., Cell. Signal. 17, 365-375, 2005; C. A. Steelman et al., FASEBJ. 20, 580-582, 2006; W. Yang et al., Cancer Res. 66, 1320-1326, 2006). Further, it was reported that myostatin is expressed at an increased level in muscular dysplasia caused by glucocorticoids (D. L. Allen et al. J. Appl. Physiol. 109, 692-701, 2010; K. Ma et al., Am. J. Physiol. 285, E363-E371, 2003), skeletal muscle degeneration-related diseases caused by HIV infections (N. F. Gonzalez-Cadavid et al., Proc. Natl. Acad. Sci. U.S.A. 95, 14938-14943, 1998), and chronic illnesses (K. A. Reardon et al., Muscle Nerve. 24, 893-899, 2001). It was known that an increased expression of myostatin is associated with metabolic disorder such as obesity, diabetes, and the like (D. S. Hittel et al., Diabetes. 58, 30-38, 2009; G. Milan et al., J. Clin. Endocrinol. Metab. 89, 2724-2727, 2004; Y. W. Chen et al., Biochem. Biophys. Res. Commun. 388, 112-116, 2009). The obesity is associated with the metabolic imbalance causing an increase in mass of adipose tissues and enhancing the resistance to insulin. It was reported that mRNA and protein of myostatin are expressed at increased levels in the human muscles with obesity and insulin resistance (D. S. Hittel et al., Diabetes. 58, 30-38, 2009; G. Milan et al., J. Clin. Endocrinol. Metab. 89, 2724-2727, 2004; J. J. Park et al., Physiol. Genomics. 27, 114-121, 2006). It was reported that myostatin-null mice in which myostatin is not expressed show a decrease in body fat mass and high-fat-induced insulin resistance (A. C. McPherron, and S. J. Lee, J. Clin. Invest. 109, 595-601. 40, 2002). Also, it was known that a mass of body fats is less increased by the high fat diet in the myostatin-null mice, compared to the wild-type mice (A. C. Dilger et al., Anim. Sci. J. 81, 586-593, 2010), and that overexpression of an inhibitory propeptide domain of myostatin suppresses obesity and insulin resistance induced by the high fat diet (B. Zhao et al., Biochem. Biophys. Res. Commun. 337, 248-255, 2005). Also, the current in vivo studies showed that the loss of myostatin functions increases the insulin sensitivity, resulting in increased glucose utilization (T. Guo et al., PloS. ONE. 4, e4937, 2009; J. J. Wilkes et al., Diabetes. 58, 1133-1143, 2009). And it was found that myostatin serves to promote consumption of glucose in muscular cells and regulate the glucose metabolism through an AMPK signaling pathway promoting the intake of glucose (Y. W. Chen et al., Int. J. Biochem. Cell. Biol. 42, 2072-2081, 2010).
That is, it was assumed that, when the myostatin mechanism is blocked, myostatin plays important roles in promoting the differentiation of muscles, preventing the obesity by blocking the differentiation into fat cells, and improving metabolic disorders such as diabetes. Therefore, the studies of myostatin inhibitors have been of importance. Up to now, a water-soluble ACVR2B-Fc fusion protein in which a myostatin receptor, ACVR2B, is fused to Fc was reported. And, it was reported that the ACVR2B-Fc fusion protein interferes with the activity of myostatin to inhibit formation of muscles by ACVR2B-Fc (Lee et al., Proc. Natl. Acad. Sci. U.S.A. 102, 1817-18122, 2005). As another attempt, there is a case showing an inhibitory effect of myostatin using follistatin which is known to bind to myostatin (Lee et al. Mol. Endocrinol. 24(10), 1 998-2008, 2010).
Meanwhile, DLK1 belonging to the notch/delta/serrate family is a transmembrane glucoprotein which is encoded by a dlk1 gene located on the chromosome 14q32, and is composed of 383 amino acids. The glucoprotein is divided into a 280-amino acid extracellular region, two 24-amino acid transmembrane regions, and a 56-amino acid intracellular region. In this case, the glucoprotein has 6 epidermal growth factor-like repeat domains, 3 N-glycosylation sites and 7 O-glycosylation sites, all of which are positioned out of the cell membrane. DLK1 is well known as a membrane protein, and also as a protein shed from the outside of the cell membrane by a tumor necrosis factor-alpha converting enzyme (TACE) to have separate functions (Yuhui Wang and Hei Sook Sul, Molecular and cellular biology. 26(14): 5421-5435, 2006).
DLK1 is found in various forms of 50 to 60 kDa by the glycosylations on the cellmembrane (Smas C M and Sul H S, Cell. 73: 725-34, 1993), and has 4 splicing variants formed by the alternative splicing (Smas C M et al., Biochemistry. 33: 9257-65, 1994). Among these, two larger variants have cleavage sites of proteolytic enzymes, and thus is cleaved by a proteolytic enzyme. TACE, to generate two water-soluble forms of 50 kDa and 25 kDa (Yuhui Wang et al., Journal of Nutrition. 136: 2953-2956, 2006).
DLK1 is also widely known as fetal antigen 1 (FA1) (Jensen C H et al., European Journal of Biochemistry. 225: 83-92, 1994) since DLK1 is expressed mainly in the embryonic tissues (Smas C M et al., Cell. 73: 725-34, 1993; Kaneta M et al., Journal of Immunology. 164: 256-64, 2000) and the placenta at a developmental stage, and particularly found in maternal serum at a high concentration. It was reported that DLK1 was also expressed in glandular cells of the pancreas (Kaneta M et al., Journal of Immunology. 164: 256-64, 2000), ovary cells, or skeletal myotubes (Floridon C et al., Differentiation. 66: 49-59, 2000). DLK1 is not expressed in most tissues after the child birth, but expressed only in certain cells such as preadipocytes (Smas C M et al., Cell. 73: 725-34, 1993), pancreatic islet cells (Carlsson C et al., Endocrinology. 138: 3940-8, 1997) thymic stromal cells (Kaneta M et al., Journal of Immunology. 164: 256-64, 2000), adrenal gland cells (Halder S K et al., Endocrinology. 139: 3316-28, 1998), and the like. Further, it was reported that DLK1 is expressed on paternal manoalleles due to the influence of methylation (Schmidt J V et al., Genes and Development. 14: 1997-2002, 2000; Takada S et al., Current Biology 10: 1135-8, 2000; Wylie A A et al, Genome Research. 10: 1711-8, 2000).
Meanwhile, the activin receptor type IIB (ACVR2B) is a protein that is encoded by an ACVR2B gene and is associated with the activin signaling mechanism. It is known that signal transduction by activin is involved in the generation or secretion of follicle-stimulating hormones (FSHs), and the regulation of menstruation cycles, and affects the cell proliferation and differentiation, and the apoptosis (Chen et al., Exp. Biol. And Med. 231(5): 534-544, 2006).
DLK1 is widely known as preadipocyte factor-1 (Pref-1) that plays a role of inhibiting differentiation of adipocytes, and its functions are the most widely studied (Smas C M et al., Cell. 73: 725-34; Villena J A et al., Hormone and Metabolic Research. 34: 664-70, 2002). Beside the ability to inhibit the differentiation of adipocytes, DLK1 is also known as it serves to inhibit the differentiation of hematopoietic stem cells (Sakajiri S et al., Leukemia. 19: 1404-10, 2005; Li L et al., Oncogene. 24: 4472-6, 2005) and regulate the differentiation of lymphoid progenitor cells (Bauer S R et al., Molecular and Cellular Biology. 18: 5247-55, 1998; Kaneta M et al., Journal of Immunology. 164: 256-64, 2000) and is involved in the wound healing (Samulewicz S J et al., Wound Repair and Regeneration. 10: 215-21, 2002). Further, it was reported that DLK1 is required for the development and regeneration of skeletal muscles (Jolena N. et al., PLoS One 5(11), e15055, 2010) and the overexpression of DLK1 causes a callipyge phenotype to generate larges muscles (Erica Davis et al., Current Biology, 14, 1858-1862, 2004).
As known so far, it can be seen that DLK1 serves to increase the muscle mass and inhibit the differentiation of adipocytes. Therefore, DLK1 has advantages over the conventional myostatin inhibitors in that it plays two important roles in inhibiting the generation of adipocytes and promoting the generation of muscular cells.
Accordingly, the present inventors have conducted ardent research to elucidate an action mechanism of an extracellular water-soluble domain of DLK1 to promote differentiation of muscular cells and inhibit differentiation of adipocytes, and found that the extracellular water-soluble domain of DLK1 binds to activin receptor type IIB(ACVR2B) serving as myostatin receptor to block binding of ACVR2B to myostatin so that it can inhibit an inhibitory effect of myostatin on muscle differentiation (i.e., myogenesis), and also directly binds to myostatin so that it can affect an inhibitory mechanism of myostatin. Therefore, the present invention has been completed based on these facts.